Secondary metabolites from Garcinia daedalanthera Pierre leaves (Clusiaceae)

Article abstract of DOI:10.1080/14786419.2020.1777117

Two new glycerol esters, (S)-2-hydroxy-3-(octanoyloxy)propyl tetracosanoate (1) and (S)-3-(((S)-11-acetoxy octadecanoyl)oxy)propane-1,2-diyl diacetate (2), and eight known compounds, docosanedioic acid (3), 2,5-dimethylnonadecane (4), lupeol (5), stigmast

Full text of DOI:10.1080/14786419.2020.1777117

Natural Product Research
Formerly Natural Product Letters
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Secondary metabolites from Garcinia
daedalanthera Pierre leaves (Clusiaceae)
Roshamur Cahyan Forestrania, Gerardo D. Anaya-Eugenio, Ulyana Muñoz
Acuña, Yulin Ren, Berna Elya & Esperanza Carcache de Blanco
To cite this article: Roshamur Cahyan Forestrania, Gerardo D. Anaya-Eugenio, Ulyana Muñoz
Acuña, Yulin Ren, Berna Elya & Esperanza Carcache de Blanco (2020): Secondary metabolites
from Garciniaꢀdaedalanthera Pierre leaves (Clusiaceae), Natural Product Research, DOI:
10.1080/14786419.2020.1777117
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1777117
Secondary metabolites from Garcinia daedalanthera
Pierre leaves (Clusiaceae)
Roshamur Cahyan Forestraniaa, Gerardo D. Anaya-Eugenioa,
a
a
b
~
~
Ulyana Munoz Acuna , Yulin Ren , Berna Elya and
Esperanza Carcache de Blancoa
aDivision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State
b
University, Columbus, OH, USA; Faculty of Pharmacy, University of Indonesia, Depok, Indonesia
ABSTRACT
ARTICLE HISTORY
Received 15 November 2019
Accepted 22 May 2020
Two new glycerol esters, (S)-2-hydroxy-3-(octanoyloxy)propyl tet-
racosanoate (1) and (S)-3-(((S)-11-acetoxy octadecanoyl)oxy)pro-
pane-1,2-diyl diacetate (2), and eight known compounds,
docosanedioic acid (3), 2,5-dimethylnonadecane (4), lupeol (5),
stigmasterol (6), b-sitosterol (7), heptadecanoic acid (8), hexane-
dioic acid, 1,6-bis[(2R)-ethylhexyl] ester (9), and 1,3-di-O-[2’,2’-di-
(p-phenylene)] (10) were isolated from the leaves of Garcinia dae-
dalanthera Pierre, collected from Indonesia. Structural analysis of
the isolates was performed using 1 D- and 2 D-NMR, LC- and GC-
MS, IR, polarimetry, and UV-visible spectroscopic methods.
Cytotoxicity assessments, as well as reactive oxygen species (ROS)
analysis of the isolates, were also completed. Lupeol was the only
compound found active with an IC50 value of 19.2mM against HT-
29 colon cancer cells. Significant ROS inhibition and induction
activity was observed for compounds 4 and 8, respectively.
KEYWORDS
Garcinia daedalanthera;
spectroscopic analysis; fatty
acid; glycerol ester; reactive
oxygen species; cytotoxicity
CONTACT Esperanza J. Carcache de Blanco
carcache-de-blan.1@osu.edu
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1777117.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
R. C. FORESTRANIA ET AL.
1. Introduction
Garcinia is a plant genus of more than 300 species that belongs to the Family
Clusiaceae (Hemshekhar et al. 2011; Koranappallil 2017). It is largely distributed in
tropical and humid regions, such as Africa, Asia, Australia, and Polynesia (Chandran
1996; Lathifah et al. 2010; Hemshekhar et al. 2011). G. daedalanthera is originated from
Sulawesi Island and also found in other rain forests in Indonesia (Najib et al. 2017). A
recent in vivo study in rats observed an antihyperlipidemic activity of an ethanolic
extract of G. daedalanthera leaves (Najib et al. 2018). Phytochemical screening
identified the following classes of compounds, sterols, saponins, alkaloids, and other
phenolic compounds (Najib et al. 2017). In a cancer-related research, the benzophe-
none garciniagifolone isolated from G. preussi was found to show cytotoxicity against
human HT-29 colon and DU-145 prostate cancer cell lines (Biloa Messi et al. 2014).
However, isolation of phytochemical constituents from G. daedalanthera and biological
activities on cancer therapy or prevention have not been reported. Therefore,
this study focus on isolation and biological evaluation of secondary metabolites
from G. daedalanthera.
Ten compounds were elucidated using 1 D- and 2 D-NMR, LC-MS and GC-MS, IR,
polarimetry, and UV-visible physical spectroscopic methods. Two new glycerol esters,
(S)-2-hydroxy-3-(octanoyloxy)propyl tetracosanoate (1) and (S)-3-(((S)-11-acetoxyoctade-
canoyl)oxy)propane-1,2-diyl diacetate (2) were structurally elucidated. Also, eight
known compounds were identified. Cytotoxicity and the potential against reactive
oxygen species (ROS) of all isolates were evaluated.
2. Results and discussion
Compound 1 was isolated as white solid. Its molecular formula assigned C35H68O5 was
deduced by HRESIMS m/z 591.4973 [M þ Na]þ (calcd for the sodiated ion C35H68O5Na
at 591.4959). ESIMS fragmentation at m/z 485.8124 was formed via McLafferty
rearrangement due to fragmentation of a fatty acid ester (Figure S7) (Fagerquist et al.
1999). A common mass fragment of a fatty acid ester was observed at m/z 443.2234.
IR showed ꢀmax bands at 3383 cmꢀ1 for hydroxyl group, at 1732 cmꢀ1 for carbonyl
ester, and at 2849 and 2917 cmꢀ1 alkane groups. 1H NMR and 13C NMR data
(Table S2) included two methyls (dH 0.86, t, H-24/80), thirty methylene (dH 2.32, t,
H-2/20; 1.60, m, H-3/30; 1.23, m, H-(4-23)/H-40/50; 1.24, m, H-60; 1.26, m, H-70; 4.15, m,
H-100/300), a methine (dH 4.09, m, H-200), and two carbonyl carbons (dC 174.16, C-1/10).
1
The molecule substitution pattern was established based on H-1H COSY correlations
from H-2 to H-3. HMBC correlations from H-100 to C-1, H-100 to C-200, and H-300 to C-100
indicated the presence of a glyceryl group attached to a carbonyl group of a fatty
acid ester. The presence of fatty acid diesters (octanoic and tetracosanoic acid esters)
attached to the glycerol group at C-300 and C-100 were derived via COSY (H-24 to H-23,
H-3 to H-2, H-80 to H-70, H-30 to H2’, H-30 to H-40) and HMBC (H-24 to C-22, H-24 to
C-23, H-23 to C-21, H-22 to C-23, H-22 to C-20, H-21 to C-19, H-6 to C-4, H-3 to C-5, H-
2 to C-4, H-3 to C-1, H-80 to C-60, H-60 to C-40, H-30 to C-10, H-20 to C-40, H-20 to C-10)
correlations. Lengths of fatty ester chains were confirmed by a hydrolysis reaction.
Analysis of fatty acid hydrolysis using EIGC-MS positive-ion mode identified fragment

NATURAL PRODUCT RESEARCH
3
ions at m/z 149 (tR 1,013 seconds) and 386 (tR 1,020 seconds), representing octanoic
and tetracosanoic acid ions, respectively. The ion at m/z 149 was derived from H2O
loss from the carboxylic acid group of a free fatty acid chain, while the fragment ion
at m/z 371 was originated from the loss of a methyl group from the tetracosanoic
acid. This fragmentation type is widely reported in saturated fatty acids (Murphy and
Axelsen 2011; Bollinger et al. 2013). Moreover, a typical McLafferty rearrangement
resulted in a fragment ion at m/z 261 (Fagerquist et al. 1999). The GC-MS fragments
at m/z 261 and 149 corresponded to the parent molecule 1 in Figure S20. Specific
25
rotation of 1 [a]D þ5 (c 0.1, MeOH) was compared with that of (S)-(þ)-propylene
25
glycerol ([a]D þ16.5, neat) (Sigma-Aldrich 2019), indicating the optical activity of
1
compound 1. H NMR signal correlations at dH 5.36 (H-200) suggested acetylation of
the hydroxyl group attached at C-200. In comparison with the chemical shift of unacety-
lated 1, the proton at C-200 after acetylation was shifted further downfield at dH 4.25
(H-300 and H-100) (Figure S18). 2 D-NOESY revealed no spatial correlation between H-200
and the methyl protons of the acetyl group, suggesting S configuration at position C-
200. Stereochemistry of compound 1 was also confirmed by depicting stable conforma-
tions via energy minimization through MM2 force field (total energy 26.1320 kcal/mol),
using Chem3D software (PerkinElmer Inc., USA). Based on these observations, com-
pound 1 was assigned as the new compound (S)-2-hydroxy-3-(octanoyloxy)propyl
tetracosanoate].
Compound 2 was isolated as colorless oil. Its molecular formula assigned C27H48O
was deduced by HRESIMS m/z 523.32384 [M þ Na]þ (calcd for 523.3247, for
C27H48O8Na). MS fragmentation at m/z 463.3033 and m/z 381.3003 indicated the loss
of the acetyl groups from the parent molecule. A similar MS fragmentation pattern as
for compound 1 was observed at m/z 265.2533, which resulted from water loss out of
the carboxylic acid group (Bollinger et al. 2013; Dunnivant and Ginsbach 2017). Typical
McLafferty fragmentation (at m/z 159.0657) occurred at the b bond between C-2 and
C-3 (Fagerquist et al. 1999; Winter 2005). IR exhibited bands of carbonyl ester and
1
alkane groups at 1747 and 2928 cmꢀ1, respectively. H NMR data (Table S2) consisted
of four methyl (dH 0.85, t, H-18; 2.06, s, H-2a; 2.07, s, H-2b; 2.01, s, H-2c), seventeen
methylene (dH 2.29, m, H-2, 1.59, m, H-3; 1.24, m, H-4/5/6/7/8/14/15/16; 1.48, m, H-10/
12; 1.25, m, H-9/13/17; 4.26, 4.14, m, H-10/30), and two methine protons (dH 4.83, m, H-
11; 5.23, m, H-20). In turn, 13C NMR data (Table S2) revealed four quaternary carbons
(dC 173.56, C-1; 170.32, C-1a; 170.73, C-1b; 171.17, C-2c). HMBC correlations suggested
the attachment of an acetyl group at C-11 via correlations from H-11 to C10, C13, C-
1
19, and C-1c (Figure S30, S31). H-1H COSY data showed correlations from H-11 to H-
10 and H-12 (Figure S28). Furthermore, the MS fragmentation ion at m/z 159.0657,
which resulted from McLafferty rearrangement, is consistent with these results. This
typical fragmentation occurred due to c-hydrogen transport from C-12 to the carbonyl
group, allowing further breakdown at the terminal double bond, C-11 and C-10
(Andersson 1978; Fagerquist et al. 1999; Mok et al. 2016). HMBC correlations from H-20
to C’3, H-30 to C-1, H-10 to C-1a and C-30, and a weak correlation between H-20 to C-1b
suggested an acetyl group at position C-2 of the glycerol group in the molecule.
An ester group was identified through HMBC correlations from H-2 to C-1, C-3 and
C-4, H-3 to C-4 and C-5, H-7 to C-5 and C-6, and H-30 to C-1. HMBC also showed

4
R. C. FORESTRANIA ET AL.
attachment of an acetyl group at position C-11 through correlations H-11 to C-13, H-
12 to C14, H-12 to C-13, H-14 to C-13, H-15 to C-13, H-16 to C14, H-15 to C-17, H-18
to C-17 and C-16. Chiral centers in compound 2 were identified by specific rotation
[a]25 þ90 (c 0.1, MeOH). Energy minimization was also obtained for this purpose
D
through MM2 force field with total energy 29.1613 kcal/mol, using Chem3D software
(PerkinElmer Inc., USA). 2 D-NOESY indicated one possible isomer (11S) at C-11 position
via correlation from dH 4.83 (H-11) to 1.48 (H-10, H-12) and dH 4.83 (H-11) to 1.25 (H-9,
H-13) (Figures S32, S33). Relative configuration at C-20, S configuration (2’S), was
inferred by NOESY correlations from dH 5.23 (H-30) to 4.14 and 4.26 (H-10, H-20). Based
on these observations, compound 2 was named as the new glycerol ester, (S)-3-(((S)-
11-acetoxy octadecanoyl)oxy)propane-1,2-diyl diacetate.
The known compounds were identified as: docosanedioic acid (3) (Korul0Kina et al.
2004), 2,5-dimethylnonadecane (4) (EPA (United States Enviromental Protecction
Agency) 2020), lupeol (5) (Jamal et al. 2008), stigmasterol (6) (Chaturvedula and
Prakash 2012), b-sitosterol (7) (Nohong et al. 2018), heptadecanoic acid (8) (NIST
2019), hexanedioic acid, 1,6-bis[(2R)-ethylhexyl] ester (9) (Li et al. 2014), and 1,3-di-O-
[20,20-di-(p-phenylene)] (10) (Qi et al. 2004), by NMR analysis and their comparison with
previous data reported in the literature. Compounds 3, 4, 8, and 9 were being
reported for the first time in the genus Garcinia (Figure 1).
Cytotoxicity of isolated compounds was assessed against DU-145, PC-3, HeLa, HT-
29, MCF-7, and MDA-MB-231 cancer cell lines. Only compound 5 exhibited cytotoxicity
towards HT-29 cells with an IC50 value of 19.2 mM (Table S1).
ROS analysis was carried out in HT-29 colon cancer cell line under two conditions,
pre-treated or untreated with ROS inducers, FeSO4 (ferrous sulfate) and H2O2 (hydro-
gen peroxide), according to previously reported protocol (Munoz Acuna et al. 2013).
Compound 2 showed ROS inhibition at 50 mM in HT-29 cells pre-treated with ROS
inducers. ROS inhibition activity towards these cells was also observed for compounds
3 and 4. Compound 4 exhibited the highest ROS inhibition against HT-29 cells
(36.12%) with p < 0.01, followed by compound 2 (10%), at 50 mM (Figure S4). This
result is in agreement with antioxidant activity reported for the same compound class,
showing radical scavenging activity at 39 and 8 mg/mL, using 2,20-azino-bis-3-ethyl-
benzthiazoline-6-sulphonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays,
respectively (Jelassi et al. 2017). Thus, compounds 2-4 indicated potential protective
activity towards ROS generation. Compound 5 at 50 mM resulted in ꢁ1.15 fold of ROS
induction, compared to daunomycin (4 mM) with ꢁ1.46 fold of ROS induction
(Figure S2). Hence, ROS activity in HT-29 cells may be associated with the cytotoxic
activity of lupeol. Mild ROS induction activity (ꢁ1.24 fold at 50 mM) was observed for
compound 6 in ROS-induced HT-29 cells (Figure S3). Compound 8 showed the highest
ability to induce ROS (ꢁ1.48 fold induction) at 0.5 mM in cells untreated with ROS
inducers (p < 0.001), when compared to the positive standard daunomycin (ꢁ1.25 fold
induction) at 0.4 mM and to the negative control. This compound exhibited ꢁ1.1 fold
ROS induction at 5 mM and daunomycin exhibited ꢁ1.3 fold ROS induction at 4 mM,
both in ROS-induced HT-29 cells (Figure S5). These results suggested similar ROS gen-
eration potency between the positive control, daunomycin, and compound 8. Hence,
heptadecanoic acid played a role in colon cancer cells redox state through ROS

NATURAL PRODUCT RESEARCH
5
Figure 1. Structures of compounds 1–10.
induction. In previous in vivo and in vitro studies, odd- and branched-chain fatty acids,
such as 13-methyl-tetradecanoic acid (iso C15:0), have shown anticancer potential
through apoptosis induction and inhibition of cancer cell proliferation (Yang et al.
2000; Vlaeminck et al. 2006). Thus, compound 8 may have potential for further studies
in cancer related research.
3. Conclusion
In this study, ten compounds were isolated from the leaves of G. daedalanthera ethyl
acetate extract. Two new glycerol esters, compounds 1 and 2, were reported from this
work. Significant ROS activity towards HT-29 cells was shown by compounds 4 and 8.
These results indicated a possible role of the isolates in mediating ROS related dis-
eases, including in cancer chemoprevention and cancer chemotherapy.

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R. C. FORESTRANIA ET AL.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This study was supported by the College of Pharmacy, Ohio State University, and the
Endowment Funds for Education (LPDP), Jakarta, Indonesia. The authors would like to acknow-
ledge Dr. Craig McElroy, College of Pharmacy, Ohio State University, for support in the use of
spectroscopic instruments.
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